Abstract
DNA sequencing analysis. Automated DNA sequencing was carried on double-stranded DNA templates by the dideoxynucleotide chain-termination method in the University of Cambridge, Department of Biochemistry, DNA sequencing facility. Taq dideoxy terminator sequencing reactions were carried out using the Big Dye Terminator kit v. 3.1 and analysed using an AB 3730XL DNA sequencer according to the manufacturer's protocols.
Cosmid DNA for initial end sequencing was prepared from overnight growth of 1 ml culture in 96-well plates in 2x LB medium containing kanamycin and ampicillin. The complete sequence of the cosmid was obtained by shotgun sequencing using Sau3AI for partial digestion of the parent cosmid. The 25 kbp size fraction of this digest was eluted from an agarose gel using the Gene Clean kit (Bio 101, Bio-Rad) and subcloned into the BamHI site of dephosphorylated pSG397 (Hashimoto-Gotoh et al., 1995). These random subclones were sequenced using template DNA obtained from 1 ml cultures grown overnight at 37 °C in 96-well plates, with universal forward and reverse sequencing primers.
PHRED (Ewing et al., 1998; Ewing & Green, 1998), PHRAP () and CONSED v14 (Gordon et al., 1998, 2004) were used for sequence basecalling, assembly and editing. The remaining gaps were finished by primer-walking sequencing, using custom-designed oligonucleotide primers with cosmid template. Both strands were completely sequenced with a mean eightfold coverage per base. Database searches were done using BLAST (Altschul et al., 1997).